BEAVERS OF THE FISH WORLD: CAN WOOD-EATING CATFISHES ACTUALLY DIGEST WOOD? A NUTRITIONAL PHYSIOLOGY APPROACH By DONOVAN PARKS GERMAN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2008 1 © 2008 Donovan Parks German 2 To my parents, John C. and Gillian R. German, for their unending support for whatever it is I choose to do in life. 3 ACKNOWLEDGMENTS I thank my advisor, David H. Evans, as well as my committee members, Karen A. Bjorndal, Douglas J. Levey, Larry M. Page, and Richard D. Miles for their invaluable support and guidance throughout this study. The academic and financial support given to me by the committee simply could not be offered by any other biology program in the country, and in that regard, the University of Florida is second to none. I am extremely indebted to the following undergraduate research assistants whose hard work and willingness to do almost anything made this project possible: Jennette Villeda, Ana Ruiz, Daniel Neuberger, Ankita Patel, Meaghan Callahan, Rosalie Bittong, Norma Lizardo, Robyn Monckton, and Joseph Taylor. Additionally, I am grateful to Alfred Thomson, Dieldrich Bermudez, Samantha Hilber, Nathan Lujan, Krista Capps, Don Taphorn, Alex Flecker, David Weineke, and Jeremy Wright who assisted with the collection and dissection of fishes. A special thank you is offered to the Barker-Emmerson family in Orlando, FL, for giving me access to their land and allowing me to collect fish from their private spring (Starbuck Spring). Pratap Pullammanappallil, Jason Curtis, Andrew Zimmerman, Lou Guillette, David Julian, and Steve Phelps graciously allowed me to conduct analyses in their laboratories or store samples in their freezers at the University of Florida. Karen Kelly, Kim Backer-Kelly, and Lynda Schneider provided indispensible support in the Electron Microscope Core Laboratory in the ICBR at the University of Florida. Brian Silliman offered his unending financial and logistic support, without which I would not have been able to function outside the university. I also thank the graduate students, post docs, and faculty in the Department of Zoology for providing intellectual and emotional support throughout my graduate career. In particular, I would like to acknowledge Jada White, Ryan McCleary, Greg Pryor, Larisa Grawe-DeSantis, Derek DeSantis, Alex Jahn, Silvia Lomascolo, Brandon Moore, Thea Edwards, James Nifong, 4 Schuyler Van Montfrans, Benjamin Predmore, and Kim Reich. I would like to thank my wonderful lab mates Kelly Hyndman, Keith Choe, Leslie Babonis, and Justin Havird for methodological and emotional support. I am extremely grateful to my wife, Lisa, and our adorable son, Merrick, who have literally kept me alive through this process. And finally, I thank my parents, John C. and Gillian R. German for supporting me in whatever I choose to do in life. Funding for this work came from the University of Florida Mentoring Opportunity Program (2), SPICE (NSF GK-12) Fellowships (2), American Museum of Natural History Theodore Roosevelt Memorial Fund, American Society of Ichthyologists and Herpetologists Raney Awards (2), Brian Reiwald Memorial Scholarships (2), The Archie Carr Center for Sea Turtle Research, NSF All Catfish Species Inventory (PI: L.M. Page), Hartz-Mountain Corporation, and the deep pockets of my advisor, David H. Evans, and my committee member Richard D. Miles. All work described in this dissertation was approved by the Institutional Animal Care and Use Committee at the University of Florida (protocols D995 and E822). 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES...........................................................................................................................8 LIST OF FIGURES .......................................................................................................................10 ABSTRACT...................................................................................................................................12 CHAPTER 1 INTRODUCTION: WHO EATS WOOD AND HOW DO THEY DO IT?..........................14 2 BEAVERS OF THE FISH WORLD: CAN WOOD-EATING CATFISHES ACTUALLY DIGEST WOOD? ............................................................................................18 Introduction.............................................................................................................................18 Materials and Methods ...........................................................................................................23 Fish Collection.................................................................................................................23 Gut Morphology and Length...........................................................................................23 Gut pH and Redox Measurements...................................................................................24 Histological and TEM Analyses......................................................................................25 Tissue Preparation for Digestive Enzyme Analyses .......................................................26 Assays of Digestive Enzyme Activity.............................................................................28 Gut Fluid Preparation, Gastrointestinal Fermentation, and Luminal Carbohydrate Profiles .........................................................................................................................33 Fiber Digestibility............................................................................................................34 Transit Time of Wood in the Digestive Tract .................................................................36 Statistical Analyses..........................................................................................................37 Results.....................................................................................................................................38 Gut Length, pH and Redox..............................................................................................38 Histology and TEM Analyses .........................................................................................39 Digestive Enzyme Activities ...........................................................................................39 Gastrointestinal Fermentation and Luminal Carbohydrate Profiles................................46 Fiber Digestibility and Gut Transit..................................................................................47 Discussion...............................................................................................................................49 3 CAN WOOD-EATING CATFISHES ASSIMILATE NUTRIENTS AND ENERGY FROM WOOD? INSIGHTS FROM STABLE ISOTOPES IN THE LABORATORY AND IN THE FIELD .............................................................................................................87 Introduction.............................................................................................................................87 Methods ..................................................................................................................................92 Fish Collection and Maintenance in Laboratory .............................................................92 Tissues Used for Stable Isotope Analysis .......................................................................93 6 Stable Isotope Trial 1: Initial Turnover...........................................................................94 Stable Isotope Trial 2: Wood Assimilation and Negative Control..................................94 Stable Isotopic Profiles of Wild-Caught Fish and Resources .........................................97 Sample Preparation for Mass Spectrometry ....................................................................98 Statistical Analyses..........................................................................................................99 Results...................................................................................................................................102 Trial One: Initial Turnover ............................................................................................102 Trial Two: Wood Assimilation and Negative Control ..................................................103 Wild-Caught Fish and Resources ..................................................................................105 Discussion.............................................................................................................................106 4 CONCLUSIONS ..................................................................................................................135 LIST OF REFERENCES.............................................................................................................139 BIOGRAPHICAL SKETCH .......................................................................................................154 7 LIST OF TABLES Table page 2-1 Digestive enzymes assayed in this study ...........................................................................64 2-2 Interspecific comparisons of body mass, relative gut length, gut length as a function of snout-vent length, and Zihler’s index in three species of loricariid catfishes. ..............65 2-3